Structure and function of the ACMV geminivirus replication initiator protein
Final Report Abstract
The replication-associated protein (Rep) of plant geminiviruses is a multifunctional protein necessary for initiation of viral replication by rolling circle replication acting as an endonuclease and for interaction with various host cell factors to modify the host cells to a favourable environment for the virus. Geminiviruses are a major threat for crop plants worldwide, and African cassava mosaic virus (ACMV) in particular is considered to be one of the ten economically most important plant viruses. The Rep protein of ACMV has been ectopically expressed in fission yeast (Schizosaccharomyces pombe) in fusion with a N-terminal GST and FLAG tag. Fusion proteins have been affinity purified employing both the GST and FLAG tags. Biochemical analyses revealed higher molecular weight complexes for the GST-Rep protein that might consist of double hexamers as it is known for other viral helicases. In contrast, FLAG-Rep is present as monomers and forms hexameric complexes. Both, GST-Rep and FLAG-Rep are active as an endonuclease for the geminiviral nonanucleotide and as a helicase. GST-Rep has been analysed with various cofactors and DNA molecules by negative stain electron microscopy. Protein complexes can be detected, but further image processing has been impeded by heterogeneity. Further experiments should address possibilities to stabilise the Rep protein and to reduce the complexity. Since the Rep protein expressed in fission yeast is a compelling model system to study interference of the Rep protein with cell cycle controls, we characterised a potential cyclin interaction motif (RXL) in the sequence of Rep that might be an alternative link to cell cycle controls to the known interactions with plant homologs of retinoblastoma protein (pRBR). Mutation of the RXL motif abrogated rereplication implying that Rep interacts via its RXL motif with one or several yeast proteins. Additionally, the RXL motif in the Rep protein was essential for viral infection in Nicotiana benthamiana plants. In order to identify new Rep interaction partners in fission yeast, we employed the BioID method and found promising candidates that have functions during the cell cycle or cell cycle control. The Rep protein expressed in fission yeast cells was immunolocalised by electron microscopy in the nucleus and cytoplasm. Although not all objectives of the project could be addressed, we obtained new insights into the Rep protein, the oligomerisation state, and how it interacts with other proteins. Further experiments are required to make the protein suitable for structural analyses.
Publications
- (2014) The RXL motif of the African cassava mosaic virus Rep protein is necessary for rereplication of yeast DNA and viral infection in plants. Virology 462-463, 189-198
Hipp, K., Rau, P., Schäfer, B., Gronenborn, B., and Jeske, H.
(See online at https://doi.org/10.1016/j.virol.2014.06.002)
